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Transcript
Mouse Pharmacokinetics (PK), Dose Fractionation and
Efficacy of TP-559 in Murine Infection Models Challenged With
Pseudomonas aeruginosa
P 1425
22nd
ECCMID
31 March – 3 April 2012
London, United Kingdom
Contact:
Leland Webster
Tetraphase Pharmaceuticals
[email protected]
J. Sutcliffe*, X.Y. Xiao, R. Clark, T. Grossman
Tetraphase Pharmaceuticals, Watertown, US
Revised Abstract
Results
Objective: To determine PK in mice and to compare the efficacy of TP-559 to comparators in
mouse models of lung and thigh infections with P. aeruginosa.
TP-559 and Comparators in the Immunocompetent Murine Lung Model Challenged with P. aeruginosa PA1145
Methods: PK evaluation of TP-559 over 24 hours (hr) was performed in CD-1 mice after 1
mg/kg IV administration using LC/MS/MS quantitation and NonWinLin. For an
immunocompetent lung infection model, female BALB/c mice (18-20) grams were infected
intranasally with P. aeruginosa PA1145. At 2 and 12 hr post-infection, mice (n = 6) were
treated intravenously (IV) with 40 mg/kg TP-559, tigecycline or amikacin; intranasal
tobramycin (40 mg/kg) was also evaluated. Compound MICs were 4, 16, 4, and 0.5 μg/ml
respectively. Further, studies with 30 mg/kg IV TP-559 fractionated over 24 hr were
performed. For all studies, mice were euthanized 24 hr post-treatment initiation and colony
forming units (CFUs) per gram lung were calculated. For the thigh model, immunocompetent
female CD-1 mice (n = 5) weighing 18-20 g were inoculated with P. aeruginosa PA694 into the
right thigh. TP-559 and meropenem (MICs, 4 and 0.13 μg/ml, respectively) were administered
at 5, 15, and 40 mg/kg IV 2 and 12 hr post-infection. At 24-hr post-infection, the muscle of the
right thigh of each mouse was harvested, homogenized, serially diluted and plated on Brain
Heart Infusion agar + 0.5% charcoal for CFU determination.
Results: TP-559 was highly efficacious in the lung infection model, providing a 3.9-log
reduction in CFUs at 40 mg/kg relative to 24-hr control. Amikacin and tobramycin reduced the
CFUs in the lung by 3.6 and 4.9 logs, respectively; tigecycline did not protect. In the
neutropenic thigh model, TP-559 at 40 and 15 mg/kg provided a 4.2 and 2.5 log CFU
reduction from the 24-hr control respectively. There was no difference in the log CFU
reduction in lung (4.42 ± 0.32) when a 30 mg/kg/day dose was given QD, BID, TID, or QID.
The PK of 1 mg/kg IV in mice produced a T½, AUC (0-t), and Cmax of approximately 5.6 hr,
1307 ng*h/mL, and 888 ng/mL, respectively.
Conclusions: TP-559 (15 mg/kg IV) had efficacy comparable to amikacin/tobramycin and
was superior to tigecycline in a P. aeruginosa murine lung infection model regardless of
regimen. TP-559 was as efficacious as meropenem in a neutropenic thigh model. The
AUC(0-t) and Cmax of TP-559 in mice were 9.8- and 1.8-fold higher than that observed with a
class comparator, 1 mg/kg IV tigecycline.
Introduction
Dose
mg/kg
TP-559
IV
Tigecycline
IV
Amikacin
IV
Tobramycin
IN
Dose
mg/kg
Dose Fractionation of 30 mg/kg IV TP-559 in the Immunocompetent
Murine Lung Model
According to the Centers for Disease Control and Prevention (CDC), the overall prevalence of
P aeruginosa infections in US hospitals is approximately 4 per 1000 discharges (0.4%). P
aeruginosa is also the fourth most commonly isolated nosocomial pathogen, accounting for
10.1% of all hospital-acquired infections. It is found on the skin of some healthy persons and
has been isolated from the throat and stool of 5% and 3% of nonhospitalized patients,
respectively. The gastrointestinal carriage rates among hospitalized patients increases to 20%
within 72 hours of admission.
Tigecycline
IV
Compound
4
16
4
0.5
These pathogens are widespread in nature, inhabiting soil, water, plants, and animals in
addition to humans. Pseudomonas aeruginosa has become an important cause of infection,
especially in patients with compromised host defense mechanisms. It is the most common
pathogen isolated from patients who have been hospitalized longer than 1 week. It is a
frequent cause of nosocomial infections such as pneumonia, urinary tract infections (UTIs),
and bacteremia. Pseudomonal infections are complicated and can be life threatening,
especially if mulitidrug- or extremel-drug resistant Thus, new drugs that are active against P.
aeruginosa would be a welcome addition to the medical armamentarium.
TP-559, BID
TP-559, TID
TP-559, QID
Vehicle
Tobramycin
IN
4
0.13
Dose
Frequency
TP-559, QD
Amikacin
IV
MIC (μg/ml)
PA1145
PA694
TP-559
Tigecycline
Amikacin
Tobramycin
Meropenem
Aggregate NNISS and EU data say that P. aeruginosa accounts for 20-30% of nosocomial
pneumonias, 10-20% of urinary tract infections, and 3-10% of bloodstream infections. For
Pseudomonas aeruginosa, high proportions of resistance to fluoroquinolones, carbapenems
and combined resistance occur, although the percentage of carbapenem-resistant P.
aeruginosa in Europe (~22%) has remained steady over the past 6 years. In terms of
multidrug resistance (MDR), the proportion of MDR (resistance to ≥3 of
piperacillin/tazobactam, ceftazidime, fluoroquinolones, aminoglycosides, and carbapenems)
averages 15% for European countries, varing from 1.9% in Denmark to 50% in Romania.
The most frequent phenotype (5.2%) is extreme drug resistant (XDR) – resistant to all 5
classes (Antimicrobial resistance surveillance in Europe, 2010, www.ecdc.europa.eu).
Tetraphase has leveraged its unique chemistry platform to find compounds with activity
against P. aeruginosa. The MIC activity and spectrum are discussed in P 1448, In vitro
potency of novel tetracyclines against Pseudomonas aeruginosa and other major Gramnegative pathogens. The MIC90 values of TP-559 and comparators for the ESKAPE
pathogens are:
TP-559
IV
Blood CFU at 26 Hours
2/6 mice had average of 1.65 x 103
4/6 mice below limit of detection (BLD)
6/6 BLD
6/6 BLD
5/6 BLD; 1 mouse had 5.00 x 104
Average CFU for 6 mice was 5.88 x 107
TP-559 and Comparators in the Immunocompetent Murine Thigh Model Challenged with P. aeruginosa PA694
MIC50/90 Values in μg/ml
E. faecalis
n=21
E. faecium
n = 14
MRSA
n = 20
ESBL+ K.
pneumoniae
(n=25)
A. baumannii
(n=29)
P.
aeruginosa
(n=96)
Enterobacter
cloacae
(n=19)
1/2
0.25/1
0.063/0.25
0.25/1
0.25/1
4/8
0.25/1
Tigecycline
0.06/0.13
≤0.016/0.063
0.13/0.13
0.5/1
0.5/4
16/32a
1/4
CXA-101
>32/>32
>32/>32
>32/>32
>16/>16
32/>32
2/4b
8/>32
Tobramycin
ND
ND
ND
ND
ND
1/>32
ND
Gentamicin
32/>32
>32/>32
1/>32
16/>32
16/>32
ND
1/>32
Meropenem
ND
ND
ND
0.063/32
2/>32
0.5/16
0.063/0.5
Levofloxacin
2/>32
>32/>32
8/>32
32/>32
4/32
1/32c
1/16
Ceftazidime
ND
ND
ND
>32/>32
ND
4/>32c
ND
Ceftriaxone
ND
>32/>32
>32/>32
>32/>32
ND
ND
32/>32
Pip/Tazo
ND
ND
ND
16/>32
ND
16/>128c
ND
Colistin
ND
ND
ND
0.25/1
0.5/1
1/1
0.25/2
1/>32
>32/>32
1/1
ND
ND
ND
ND
Compound
TP-559
Vancomycin
a
55 P. aeruginosa isolates; b 20 P. aeruginosa isolates; c 76 P. aeruginosa isolates; ND, not done
Methods
Susceptibility. Using CLSI methodology, the MICs of TP-559, tigecycline, amikacin, and
tobramycin against P. aeruginosa PA1145 (challenge strain for lung model) and TP-559 and
meropenem for P. aeruginosa PA694 (challenge strain for thigh model) were determined (see
Table). PA1145 is from the VivoSource (Waltham, MA) strain collection and was originally
obtained as a cystic fibrosis isolate from Children’s Hospital, Boston. PA694 was obtained from
Eurofins-Medinet and is a 2009 urine isolate from the US. The latter strain was identified from a
collection of isolates screened for reproducible infection in the thigh model by the University of
North Texas Health Science Center.
Dose
mg./kg
Lung infection model. Immunocompetent female BALB/c mice (18-20 grams) were infected
intranasally (0.05 ml under light anesthesia) with ~2 x 107 CFU PA1145/mouse. At 2 (CFU
burden not significantly different than inoculum) and 12 hr post-infection, mice (n = 6) were
treated intravenously (IV) with 40 mg/kg tigecycline or amikacin. TP-559 was administered IV at
doses of 0.5-15 mg/kg. Tobramycin (40 mg/kg) was evaluated intranasally. Dose fractionation
studies over 24 hours were performed with 30 mg/kg IV TP-559 in groups of 6 mice. The lungs of
the mice were aseptically removed, weighed, homogenized, serially diluted, and plated on
MacConkey media. Blood was aseptically collected at 26 hours into heparin tubes and provided
to the Sponsor for culturing. The plates were incubated overnight at 37C in 5% CO2. CFU per
gram of lung was calculated by enumerating the plated colonies then adjusting for serial dilutions
and the weight of the lung. For all studies, mice were euthanized 24 hr post-treatment initiation
and colony forming units (CFUs) per gram lung were calculated. The untreated controls die
between 26-36 hrs post-challenge, having a 1.5 – 2 log CFU lung burden than when therapy was
initiated). Occasionally, death occured as early as 20-24 hrs.
Pharmacokinetics of 1 mg/kg TP-559 and Tigecycline Administered IV in CD-1 Mice
Thigh infection model. Immunocompetent female specific-pathogen-free CD-1 mice (n = 5)
weighing 22 ±2 g were inoculated intramuscularly (0.1 ml/thigh) with 3-5 x 106 CFU/mouse of
PA694 into right thigh. One group did not receive drug treatment and their thighs were harvested
at 2 hour post-infection, showing a bacterial burden per gram thigh not greatly different than
inoculum. The remaining mice were administered either TP-559 and meropenem at 5, 15, and
40 mg/kg IV 2 and 12 hr post-infection. At 24-hr post-infection, the muscle of the right thigh of
each mouse was aseptically removed and homogenized in 2 ml of PBS, pH 7.4 with a Polytron
tissue homogenizer. The bacterial burden was generally 108 – 109 CFU/gram thigh. The
homogenates were serially diluted and plated on Brain Heart Infusion agar + 0.5% charcoal (w/v)
for CFU determination. Death in mice with >109 CFU/gram thigh did occur and was also seen on
occasions when the bacterial burden was 108 CFU/gram thigh.
PK determination. CD-1 mice from Charles River Laboratories were allowed to acclimate for two
days prior to dosing. Forty-eight animals were selected for the study based on acceptable health.
Study animals were placed into two groups of twenty-four animals each. A single dose was
administered to each mouse by intravenous injection via the tail vein. Body weight was
measured prior to dosing and mice were dosed at a dose volume of 5 ml/kg based on individual
body weight. Study animals were not fasted. Mice were euthanized using CO2 and blood
samples were collected at sacrifice by cardiac puncture at the eight PK time points (2 min, 30
min, 1 hr, 2 hr, 4 hr, 6 hr, 12 hr, 24 hr). All time points were collected within 5% of the target time.
Blood collection (approximately 250 µL at sacrifice) was into pre-chilled (0 – 4ºC) heparincontaining blood collection tubes. The collected plasma (approximately 100 µL) was placed in
sample tubes, snap frozen on dry ice and immediately stored at nominally -80C. Frozen plasma
samples and dosing solution retains were analyzed for TP-599 concentration by LC/MS/MS using
an internal standard. Quality control (QC) samples (low, medium, high; minimum of 6 standards
with LLOQ < 3 ng/mL) and standard curves (in duplicate) were included in the bioanalytical run.
WinNonLin was used to determine individual and mean PK parameters ( Cmax, Tmax, T½, CL,
Vss, AUC(0-t), AUC(0-)).
TP-559
IV
Meropenem
IV
TP-559
IV
Meropenem
IV
PK Parameter @ 1 mg/kg IV
Compound
TP-559
Tigecycline
AUC0-t
(ng·h/mL)
1307
133
AUCinf
(ng·h/mL)
1418
148
T½ (h)
C0 (ng/mL)
Vss (L/kg)
5.62
2.51
888
496
5
12.7
CL
(mL/min/kg)
12
112
Conclusions
TP-559 is one of the first examples of a novel tetracycline that has efficacy
comparable to standard-of-care antibiotics in mouse lung and thigh abscess
models challenged with P. aeruginosa
TP-559 exemplifies the success of the fully synthetic tetracycline platform in
extending spectrum and retaining potency against troublesome hospital
pathogens